Physical fitness decline and career paths: a longitudinal study of medical undergraduates.

INTRODUCTION: Exercise enhances one's health and competitiveness. A strong physical fitness status can pave the way for a promising future. This study presents the time-based trends in physical fitness indicators-including height, weight, BMI, lung capacity, dash, long-distance running, and standing long jump-among medical undergraduates during their university years. Additionally, we analyzed the impact of students' physical fitness on their career paths. METHOD: We conducted a retrospective database study by collecting physical fitness test data and career paths information for 634 medical students from a university in southwestern China. These students graduated in 2022. The career paths included pursuits in further studies, employment, and unemployment. To detect differences in these aspects, we used the t-test and Chi-square test. RESULTS: Our study indicates a significant declining trend in the physical fitness of medical students during their university years. The changes observed between the first and fourth tests are as follows: Weight (kg): 58.52 ± 10.48 to 60.73 ± 12.07, P < 0.00 BMI (kg/m^2): 20.79 ± 2.74 to 21.24 ± 3.06, P < 0.00 50-m dash (s): 8.91 ± 0.99 to 9.25 ± 1.11, P < 0.00 Standing long jump (cm): 187.74 ± 30.98 to 182.59 ± 32.25, P < 0.00 800-m run for females (min): 3.84 ± 0.47 to 4.48 ± 0.85, P < 0.00 1000-m run for males (min): 3.98 ± 0.63 to 4.62 ± 0.87, P < 0.00 Sit-ups for females (count): 30.39 ± 7.5 to 29.03 ± 8.82, P < 0.00 Upon analyzing the correlation between changes in physical fitness and career paths, students with stable or decreased BMI had better post-graduation outcomes compared to students with increased BMI. CONCLUSIONS: Medical students show a declining trend in physical fitness during their undergraduate years. A good physical health status is beneficial for achieving better career paths. Medical students should place greater emphasis on physical exercise during their time in school.

One-step in vivo gene knock-out in porcine embryos using recombinant adeno-associated viruses.

Introduction: Gene-edited pigs have become prominent models for studying human disease mechanisms, gene therapy, and xenotransplantation. CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated 9 (CRISPR/Cas9) technology is a widely employed tool for generating gene-edited pigs. Nevertheless, delivering CRISPR/Cas9 to pre-implantation embryos has traditionally posed challenges due to its reliance on intricate micromanipulation equipment and specialized techniques, resulting in high costs and time-consuming procedures. This study aims to introduce a novel one-step approach for generating genetically modified pigs by transducing CRISPR/Cas9 components into pre-implantation porcine embryos through oviductal injection of recombinant adeno-associated viruses (rAAV). Methods: We first used rAAV-1, rAAV-6, rAAV-8, rAAV-9 expressing EGFP to screen for rAAV serotypes that efficiently target porcine embryos, and then, to achieve efficient expression of CRISPR/Cas9 in vivo for a short period, we packaged sgRNAs targeting the GHR genes to self-complementary adeno-associated virus (scAAV), and Cas9 proteins to single-stranded adeno-associated virus (ssAAV). The efficiency of porcine embryos -based editing was then validated in vitro. The feasibility of this one-step method to produce gene-edited pigs using rAAV-CRISPR/Cas9 oviductal injection into sows within 24 h of conception was then validated. Results: Our research firstly establishes the efficient delivery of CRISPR/Cas9 to pig zygotes, both in vivo and in vitro, using rAAV6. Successful gene editing in pigs was achieved through oviductal injection of rAAV-CRISPR/Cas9. Conclusion: This method circumvents the intricate procedures involved in in vitro embryo manipulation and embryo transfers, providing a straightforward and cost-effective approach for the production of gene-edited pigs.

Targeted Integration of siRNA against Porcine Cytomegalovirus (PCMV) Enhances the Resistance of Porcine Cells to PCMV.

In the world's first pig-to-human cardiac cytomegalovirus (PCMV), xenotransplant and elevated levels of porcine key factors contributing to patient mortality were considered. This has renewed attention on PCMV, a virus widely prevalent in pigs. Currently, there are no effective drugs or vaccines targeting PCMV, and its high detection difficulty poses challenges for prevention and control research. In this study, antiviral small hairpin RNA (shRNA) was selected and inserted into the Rosa26 and miR-17-92 loci of pigs via a CRISPR/Cas9-mediated knock-in strategy. Further in vitro viral challenge experiments demonstrated that these genetically edited pig cells could effectively limit PCMV replication. Through this process, we constructed a PCMV-infected cell model, validated partial viral interference sites, enhanced gene knock-in efficiency, performed gene editing at two different gene loci, and ultimately demonstrated that RNA interference (RNAi) technology combined with CRISPR/Cas9 has the potential to generate pig cells with enhanced antiviral infection capabilities. This opens up possibilities for the future production of pig populations with antiviral functionalities.

Visual screening of CRISPR/Cas9 editing efficiency based on micropattern arrays for editing porcine cells.

CRISPR/Cas9 technology, combined with somatic cell nuclear transplantation (SCNT), represents the primary approach to generating gene-edited pigs. The inefficiency in acquiring gene-edited nuclear donors is attributed to low editing and delivery efficiency, both closely linked to the selection of CRISPR/Cas9 forms. However, there is currently no direct method to evaluate the efficiency of CRISPR/Cas9 editing in porcine genomes. A platform based on fluorescence reporting signals and micropattern arrays was developed in this study, to visually assess the efficiency of gene editing. The optimal specifications for culturing porcine cells, determined by the quantity and state of cells grown on micropattern arrays, were a diameter of 200 µm and a spacing of 150 µm. By visualizing the area of fluorescence loss and measuring the gray value of the micropattern arrays, it was quickly determined that the mRNA form targeting porcine cells exhibited the highest editing efficiency compared to DNA and Ribonucleoprotein (RNP) forms of CRISPR/Cas9. Subsequently, four homozygotes of the ß4GalNT2 gene knockout were successfully obtained through the mRNA form, laying the groundwork for the subsequent generation of gene-edited pigs. This platform facilitates a quick, simple, and effective evaluation of gene knockout efficiency. Additionally, it holds significant potential for swiftly testing novel gene editing tools, assessing delivery methods, and tailoring evaluation platforms for various cell types.

Construction and applications of the EOMA spheroid model of Kaposiform hemangioendothelioma.

BACKGROUND: Kaposiform hemangioendothelioma (KHE) is a rare intermediate vascular tumor with unclear pathogenesis. Recently, three dimensional (3D) cell spheroids and organoids have played an indispensable role in the study of many diseases, such as infantile hemangioma and non-involuting congenital hemangiomas. However, few research on KHE are based on the 3D model. This study aims to evaluate the 3D superiority, the similarity with KHE and the ability of drug evaluation of EOMA spheroids as an in vitro 3D KHE model. RESULTS: After two days, relatively uniform morphology and high viability of EOMA spheroids were generated by the rotating cell culture system (RCCS). Through transcriptome analysis, compared with 2D EOMA cells, focal adhesion-related genes such as Itgb4, Flt1, VEGFC, TNXB, LAMA3, VWF, and VEGFD were upregulated in EOMA spheroids. Meanwhile, the EOMA spheroids injected into the subcutaneous showed more obvious KMP than 2D EOMA cells. Furthermore, EOMA spheroids possessed the similar characteristics to the KHE tissues and subcutaneous tumors, such as diagnostic markers (CD31 and LYVE-1), cell proliferation (Ki67), hypoxia (HIF-1α) and cell adhesion (E-cadherin and N-cadherin). Based on the EOMA spheroid model, we discovered that sirolimus, the first-line drug for treating KHE, could inhibit EOMA cell proliferation and downregulate the VEGFC expression. Through the extra addition of VEGFC, the effect of sirolimus on EOMA spheroid could be weakened. CONCLUSION: With a high degree of similarity of the KHE, 3D EOMA spheroids generated by the RCCS can be used as a in vitro model for basic researches of KHE, generating subcutaneous tumors and drug screening.

Tissue-specific micropattern array chips fabricated via decellularized ECM for 3D cell culture.

Multicellular three-dimensional (3D) in vitro models, such as cell spheroids and organoids, can significantly improve the viability, histomorphology, genotype stability, function and drug metabolism of cells [1], [2], [3]. In general, several culture methods of 3D models, including the hanging drop, microwell-mesh and hydrogel encapsulating methods, have difficulty building a standard mode and controlling the size and arrangement of cell spheroids or organoids, which could severely affect the authenticity and repeatability of experimental results [4]. Another key factor in 3D in vitro models is the extracellular matrix (ECM), which can determine cell viability, proliferation, differentiation, function, migration and organization [5]. In this study, micropattern array chips combined with decellularized ECM (dECM) not only provide tissue-specific ECM but also control the size and arrangement of 3D models. •Methods have been established to demonstrate the use of dECM as a bioink to generate dECM-coated micropattern array chips by microcontact printing.•The micropattern can limit cell growth and migration, and cells spontaneously assemble into cell spheroids with uniform size and orderly arrangement.

Substitutional doping of MoTe2/ZrS2 heterostructures for sustainable energy related applications.

Stacking and/or substitutional doping are effective strategies to tune two-dimensional materials with desired properties, greatly extending the applications of the pristine materials. Here, by employing first-principles calculations, we propose that a pristine MoTe2/ZrS2 heterostructure is a distinguished lithium-ion battery anode material with a low Li diffusion barrier (∼0.26 eV), and it has a high maximum Li storage capacity (476.36 mA h g-1) and a relatively low open-circuit voltage (0.16 V) at Li4/MoTe2/Li/ZrS2/Li4. The other heterostructures with different types can be achieved by substitutional doping and their potential applications in sustainable energy related areas are further unraveled. For instance, a type-II TeMoSe/ZrS2 heterostructure could be a potential direct Z-scheme photocatalyst for water splitting with a high solar-to-hydrogen conversion efficiency of 17.62%. The TeMoSe/SZrO heterostructure is predicted to be a potential candidate for application in highly efficient solar cells. Its maximum power conversion efficiency can be as high as 19.21%, which is quite promising for commercial applications. The present results will shed light on the sustainable energy applications of pristine or doped MoTe2/ZrS2 heterostructures in the future.

The prevention strategies of swine viruses related to xenotransplantation.

Xenotransplantation is considered a solution for the shortage of organs, and pigs play an indispensable role as donors in xenotransplantation. The biosecurity of pigs, especially the zoonotic viruses carried by pigs, has attracted attention. This review introduces several viruses, including porcine endogenous retroviruses that are integrated into the pig genome in a DNA form, herpesviruses that have been proven to clearly affect recipient survival time in previous xenotransplant surgeries, the zoonotic hepatitis E virus, and the widely distributed porcine circoviruses. The detail virus information, such as structure, caused diseases, transmission pathways, and epidemiology was introduced in the current review. Diagnostic and control measures for these viruses, including detection sites and methods, vaccines, RNA interference, antiviral pigs, farm biosecurity, and drugs, are discussed. The challenges faced, including those posed by other viruses and newly emerged viruses, and the challenges brought by the modes of transmission of the viruses are also summarized.

Overexpression of SPHK1 associated with targeted therapy resistance in predicting poor prognosis in renal cell carcinoma.

Background: Sphingosine kinase 1 (SPHK1) is a key enzyme that catalyzes the phosphorylation of sphingosine. Recent studies reported SPHK1 to be associated with renal cell carcinoma (RCC) progression by inducing targeted therapy resistance. However, the expression and the clinical significance of SPHK1 on RCC in those having received targeted therapy have not been elucidated. The present study explored the expression of SPHK1 in RCC tissues from targeted therapy recipients, the correlation of SPHK1 with clinicopathological parameters, and the effect of SPHK1 on RCC patient prognosis. Methods: Differential gene expression analysis of RCC treated with and without targeted therapy was performed. The correlations of SPHK1 expression with clinical parameters of RCC were examined. Gene set enrichment analysis (GSEA) was performed to clarify the potential role of SPHK1 associated with targeted therapy resistance. The value of SPHK1 as a diagnostic marker for RCC was also evaluated. The Kaplan-Meier method was applied to analyze the correlation between SPHK1 expression and patient survival rate by using the clinical data from patients with RCC. Results: Significant overexpression of SPHK1 was detected in RCC treated with targeted therapy. SPHK1 expression was closely correlated with RCC progression-related clinicopathological parameters. Therefore, elevated SPHK1 could effectively diagnose RCC and distinguish RCC with an advanced clinical stage and a high pathological grade. SPHK1 was associated with the stemness of RCC cells via the activation of the Wnt, Hedgehog, or Notch signaling pathways in targeted drug-treated or untreated RCC. Survival analysis of a large cohort of RCC samples indicated overexpression of SPHK1 to be inversely correlated with the overall and disease-free survival of patients with RCC. Conclusions: Our study indicated that SPHK1 associated with targeted therapy resistance could serve as a potential prognostic marker and a valuable biomarker of response to angiogenic agents in RCC.

The CRISPR/Cas9 System Delivered by Extracellular Vesicles.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems can precisely manipulate DNA sequences to change the characteristics of cells and organs, which has potential in the mechanistic research on genes and the treatment of diseases. However, clinical applications are restricted by the lack of safe, targeted and effective delivery vectors. Extracellular vesicles (EVs) are an attractive delivery platform for CRISPR/Cas9. Compared with viral and other vectors, EVs present several advantages, including safety, protection, capacity, penetrating ability, targeting ability and potential for modification. Consequently, EVs are profitably used to deliver the CRISPR/Cas9 in vivo. In this review, the advantages and disadvantages of the delivery form and vectors of the CRISPR/Cas9 are concluded. The favorable traits of EVs as vectors, such as the innate characteristics, physiological and pathological functions, safety and targeting ability of EVs, are summarized. Furthermore, in terms of the delivery of the CRISPR/Cas9 by EVs, EV sources and isolation strategies, the delivery form and loading methods of the CRISPR/Cas9 and applications have been concluded and discussed. Finally, this review provides future directions of EVs as vectors of the CRISPR/Cas9 system in clinical applications, such as the safety, capacity, consistent quality, yield and targeting ability of EVs.

Nootkatone Improves Chronic Unpredictable Mild Stress-Induced Depressive-Like Behaviors by Repressing NF-κB/NLRP3-Mediated Neuroinflammation.

OBJECTIVE: To explore the effect of nootkatone (NKT) on chronic unpredictable mild stress (CUMS)-induced depressive-like behaviors and the mechanism underlying NKT improving the depressive-like behaviors. METHODS: The CUMS-induced depression model was established in mice. Fifty mice were randomized into 5 groups (n=10) in accordance with a random number table: control group, CUMS group, CUMS + NKT (6 mg/kg) group, CUMS + NKT (12 mg/kg) group, and CUMS + ketamine group. From the 22th day, NKT (6 or 12 mg/kg) or ketamine (0.5 mg/kg) was given with intragastric administration every day for 21 days. Behavioral tests including forced swimming test (FST), tail suspension test (TST), sucrose preference test (SPT) and open-field test (OFT) were carried out. The mRNA and protein expressions of interleukin (IL)-1ß, IL-18, IL-6, and tumor necrosis factor (TNF)-α in hippocampus were assessed using quantitative realtime polymerase chain reaction (PCR), Western blot analysis, and enzyme linked immunosorbent assay. The nuclear factor-κB (NF-κB)/NOD-like receptor 3 (NLRP3) inflammasome pathway was analyzed using Western blot and immunofluorescence analysis. RESULTS: NKT treatment improved CUMS-induced depressive-like behaviors in mice (P<0.05 or P<0.01). NKT significantly decreased the mRNA and protein levels of IL-1ß, IL-18, IL-6, and TNF-α in hippocampus of CUMS mice (P<0.05 or P<0.01). Furthermore, NKT repressed CUMS-induced activation of NF-κB signaling and NLRP3 inflammasome (P<0.01). More important, Nigericin, a NLRP3 activator, destroyed the effect of NKT on repressing neuroinflammation and improving depressive-like behaviors (P<0.05 or P<0.01). CONCLUSION: NKT ameliorates the depressive-like symptoms, in part by repressing NF-κB/NLRP3-mediated neuroinflammation.

Three-Dimensional Microtumor Formation of Infantile Hemangioma-Derived Endothelial Cells for Mechanistic Exploration and Drug Screening.

Infantile hemangioma (IH) is the most prevalent type of vascular tumor in infants. The pathophysiology of IH is unknown. The tissue structure and physiology of two-dimensional cell cultures differ greatly from those in vivo, and spontaneous regression often occurs during tumor formation in nude mice and has severely limited research into the pathogenesis and development of IH. By decellularizing porcine aorta, we attempted to obtain vascular-specific extracellular matrix as the bioink for fabricating micropattern arrays of varying diameters via microcontact printing. We then constructed IH-derived CD31+ hemangioma endothelial cell three-dimensional microtumor models. The vascular-specific and decellularized extracellular matrix was suitable for the growth of infantile hemangioma-derived endothelial cells. The KEGG signaling pathway analysis revealed enrichment primarily in stem cell pluripotency, RAS, and PI3KAkt compared to the two-dimensional cell model according to RNA sequencing. Propranolol, the first-line medication for IH, was also used to test the model's applicability. We also found that metformin had some impact on the condition. The three-dimensional microtumor models of CD31+ hemangioma endothelial cells were more robust and efficient experimental models for IH mechanistic exploration and drug screening.

[Effects of Cas9 expression on cell growth and production of natural products in Saccharomyces cerevisiae and optimization of CRISPR-Cas9 editing system].

CRISPR-Cas9 gene editing technology has been widely used in Saccharomyces cerevisiae.However, the effects of Cas9, as an exogenous protein, on the growth and production of natural products in S.cerevisiae are still unclear.In this study, Cas9 gene was expressed in S.cerevisiae by integration into the genome and construction into vectors, and two natural products, carotenoid and miltiradiene, were selected as the target products to study the effects of Cas9 expression on yeast growth and production capacity.The results showed that whether Cas9 was integrated into the genome or expressed by vectors, Cas9 inhibited the growth of S.cerevisiae, which was more obvious in the form of genome integration.When Cas9 was integrated into the genome, it had no effect on the production of carotenoid and miltiradiene by S.cerevisiae, but when Cas9 was expressed by vectors, the ability of S.cerevisiae to produce carotenoids and miltiradiene was significantly reduced.Therefore, in order to further efficiently knock out Cas9 after gene editing and minimize the adverse impact of Ura3 and Trp1 vectors, this study systematically explored the removal efficiency of the two vectors, and a plasmid capable of efficient gene editing was constructed, which optimized the application of CRISPR-Cas9 gene editing system in S.cerevisiae, and provided reference for the application of gene editing technology based on Cas9.

Application of deep learning to identify ductal carcinoma in situ and microinvasion of the breast using ultrasound imaging.

Background: The treatment and prognosis of breast ductal carcinoma in situ (DCIS) with and without microinvasion (MIC) are different. Ultrasound imaging shows that DCIS is a heterogeneous breast tumor with diverse manifestations. DCIS means that the cancer cells are confined in the duct without penetrating the basement membrane, MIC means that the cancer cells penetrate the basement membrane and the maximum diameter of any largest invasive lesion is less than or equal to 1 mm. This study was designed to evaluate how deep learning can be used to identify DCIS with MIC on ultrasound images. Methods: The clinical and ultrasound data of 467 consecutive inpatients diagnosed with DCIS (213 with MIC) in West China Hospital of Sichuan University were collected from January 2013 to April 2019 and randomly apportioned to training and internal validation sets. An external validation set comprised data from Sichuan Provincial People's Hospital with 101 patients (33 with MIC) collected between January 2017 and December 2019. There were 2,492 original images; 66% of these were used to establish a model, and the remaining 34% were used to evaluate the model. Three experienced breast ultrasound clinicians analyzed the ultrasound images to establish a logistic regression model. Finally, the logistic regression model and five deep learning models (ResNet-50, ResNet-101, DenseNet-161, DenseNet-169, and Inception-v3) were compared and evaluated to assess their diagnostic efficiency when identifying MIC based on ultrasound image data. Results: The characteristics of high nuclear grade (P<0.001), necrosis (P=0.006), estrogen receptor negative (ER-; P=0.003), progesterone receptor negative (PR-; P=0.001), human epidermal growth factor receptor 2 positive (HER2+; P=0.034), lymphatic metastasis (P=0.008), and calcification (P<0.001) all showed significant correlations with MIC. The Inception-v3 model achieved the best performance (P<0.05) in MIC identification. The area under the receiver operating curve (AUC) of the Inception-v3 model was 0.803 [95% confidence interval (CI): 0.709 to 0.878], with a classification accuracy of 0.766, a sensitivity of 0.767, and a specificity of 0.765. Conclusions: Deep learning can be used to identify MIC of breast DCIS from ultrasound images. Models based on Inception-v3 can provide automated detection of DCIS with MIC from ultrasound images.

Autoclaving pHEMA-Based Hydrogels Immersed in Deionized Water has No Effect on Physicochemical Properties and Cell Behaviors.

Hydrogels based on poly-(2-hydroxyethyl methacrylate) (pHEMA) have been widely used as biomaterials in tissue engineering due to their biocompatibility, hydrophilicity, and low friction coefficient. The terminal sterilization of hydrogels is a critical step in clinical applications. However, regulations and standardization for the sterilization of hydrogels based on pHEMA are still lacking. In this study, we explored six sterilization methods on pHEMA-based materials (A1: pHEMA, A2: pHEMA copolymerizes with acrylic acid, and A3: pHEMA copolymerizes with acrylic acid and further coordinated with iron ions), such as gamma irradiation, 75% ethanol, ultraviolet (UV), ethylene oxide (EtO), and autoclaving with or without deionized water (autoclaving-H2O or autoclaving-dry). Combining results from the multifaceted approaches with assessment, pHEMA-based hydrogels can be completely sterilized via the autoclaving-H2O method analyzed by sterilized testing. The physicochemical properties and cell behavior of sterilized hydrogels were not influenced by this sterilization approach, validated by Fourier transform infrared (FT-IR) spectroscopy and tensile tests. The pHEMA-based hydrogel sterilized by the autoclaving-H2O method also had no effect on the cell behavior evaluated by in vitro cytotoxicity experiments and caused no evident inflammatory reaction in tissue in vivo implantation experiments. However, it was also found that there were still some defects in the A2 and A3 groups as biomaterials possibly because of an inappropriate proportion of formulations or raw material used in exploring sterilization methods. These findings have implications for the improvement and clinical application of pHEMA-based hydrogels.

Kidney ECM Pregel Nanoarchitectonics for Microarrays to Accelerate Harvesting Gene-Edited Porcine Primary Monoclonal Spheres.

One of the key steps of using CRISPR/Cas9 to obtain gene-edited cells used in generating gene-edited animals combined with somatic cell nuclear transplantation (SCNT) is to harvest monoclonal cells with genetic modifications. However, primary cells used as nuclear donors always grow slowly and fragile after a series of gene-editing operations. The extracellular matrix (ECM) formulated directly from different organs comprises complex proteins and growth factors that can improve and regulate the cellular functions of primary cells. Herein, sodium lauryl ether sulfate (SLES) detergent was first used to perfuse porcine kidney ECM, and the biological properties of the kidney ECM were optimized. Then, we used a porcine kidney ECM pregel to pattern the microarray and developed a novel strategy to shorten the time of obtaining gene-edited monoclonal cell spheroids with low damage in batches. Our results showed that the SLES-perfused porcine kidney ECM pregel displayed superior biological activities in releasing growth factors and promoting cell proliferation. Finally, combined with microarray technology, we quickly obtained monoclonal cells in good condition, and the cells used as nuclear donors to construct recombinant embryos showed a significantly higher success rate than those of the traditional method. We further successfully produced genetically edited pigs.

Differential MC5R loss in whales and manatees reveals convergent evolution to the marine environment.

Melanocortin 5 receptor (MC5R), which is expressed in the terminally differentiated sebaceous gland, is a G protein-coupled receptor (GPCR). MC5R exists mostly in mammals but is completely lost in whales; only the relic of MC5R can be detected in manatees, and phenotypically, they have lost sebaceous glands. Interestingly, whales and manatees are both aquatic mammals but have no immediate common ancestors. The loss of MC5R and sebaceous glands in whales and manatees is likely to be a result of convergent evolution. Here, we find that MC5R in whales and manatees are lost by two different mechanisms. Homologous recombination of MC5R in manatees and the insertion of reverse transcriptase in whales lead to the gene loss, respectively. On one hand, in manatees, there are two "TTATC" sequences flanking MC5R, and homologous recombination of the segments between the two "TTATC" sequences resulted in the partial loss of the sequence of MC5R. On the other hand, in whales, reverse transcriptase inserts between MC2R and RNMT on the chromosome led to the loss of MC5R. Based on these two different mechanisms for gene loss in whales and manatees, we finally concluded that MC5R loss might be the result of convergent evolution to the marine environment, and we explored the impact on biological function that is significant to environmental adaptation.

Ordered micropattern arrays fabricated by lung-derived dECM hydrogels for chemotherapeutic drug screening.

Aims: This study aims to evaluate ECM-coated micropattern arrays derived from decellularization of native porcine lungs as a novel three-dimensional cell culture platform. Methods: ECM derived from decellularization of native porcine lungs was exploited to prepare hydrogels. Then, dECM-coated micropattern arrays were fabricated at four different diameters (50, 100, 150 and 200 â€‹µm) using polydimethylsiloxane (PDMS). Two lung cancer cell lines, A549 and H1299, were tested on a dECM-coated micropattern array as a novel culture platform for cell adhesion, distribution, proliferation, viability, phenotype expression, and drug screening to evaluate the cytotoxicity of paclitaxel, doxorubicin and cisplatin. Results: The ECM derived from decellularization of native porcine lungs supported cell adhesion, distribution, viability and proliferation better than collagen I and Matrigel as the coated matrix on the surface. Moreover, the optimal diameter of the micropattern arrays was 100-150 â€‹µm, as determined by measuring the morphology, viability, proliferation and phenotype of the cancer cell spheroids. Cell spheroids of A549 and H1299 on dECM-coated micropattern arrays showed chemoresistance to anticancer drugs compared to that of the monolayer. The different distributions of HIF-1α, MCL-1 (in the center) and Ki-67 and MRP2 (in the periphery) of the spheroids demonstrated the good establishment of basal-lateral polarity and explained the chemoresistance phenomenon of spheroids. Conclusions: This novel three-dimensional cell culture platform is stable and reliable for anticancer drug testing. Drug screening in dECM-coated micropattern arrays provides a powerful alternative to existing methods for drug testing and metabolic profiling in the drug discovery process.